Erythropoiesis is the production of red blood cells which occurs to offset cell destruction. Erythropoiesis is a controlled physiological mechanism that enables sufficient red blood cells to be available for proper tissue oxygenation. Naturally occurring human erythropoietin (hEPO) is a glycoprotein containing 165 amino acids that is produced in the kidney and is the humoral plasma factor which stimulates red blood cell production (Carnot, P and Deflandre, C (1906) C.R. Acad. Sci. 143: 432; Erslev, A J (1953 Blood 8: 349; Reissmann, K R (1950) Blood 5: 372; Jacobson, L O, Goldwasser, E, Freid, W and Plzak, L F (1957) Nature 179: 6331-4). Human EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow. Human EPO exerts its biological activity by binding to receptors on erythroid precursors (Krantz, B S (1991) Blood 77: 419). Naturally occurring human erythropoietin is an acidic glycoprotein present in low concentrations in plasma to stimulate replacement of red blood cells which are lost through aging.
Erythropoietin has been manufactured biosynthetically using recombinant DNA technology (Egrie, J C, Strickland, T W, Lane, J et al. (1986) Immunobiol. 72: 213-224) and is the product of a cloned human EPO gene inserted into and expressed in the ovarian tissue cells of the chinese hamster (CHO cells). Naturally occurring human EPO is first translated to a 166 aa containing polypeptide chain with arginine 166. In a postranslational modification arginine 166 is cleaved by a carboxypeptidase. The primary structure of human EPO (165 aa) is illustrated in FIG. 1 (SEQ ID NO:1). The primary structure of human EPO (166 aa) is illustrated in FIG. 2 (SEQ ID NO:2). There are two disulfide bridges between Cys7-Cys161 and Cys29-Cys33. The molecular weight of the polypeptide chain of human EPO without the sugar moieties is 18,236 Da. In the intact EPO molecule, approximately 40% of the molecular weight is accounted for by the carbohydrate groups (Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262: 12059).
Because erythropoietin is essential in red blood cell formation, the hormone is useful in the treatment of blood disorders characterized by low or defective red blood cell production. Clinically, EPO is used in the treatment of for example anemia in chronic renal failure patients (CRF) (Eschbach, J W, Egri, J C, Downing, M R et al. (1987) NEJM 316: 73-78; Eschbach, J W, Abdulhadi, M H, Browne, J K et al. (1989) Ann. Intern. Med. 111: 992; Egrie, J C, Eschbach, J W, McGuire, T, Adamson, J W (1988) Kidney Intl. 33: 262; Lim, V S, Degowin, R L, Zavala, D et al. (1989) Ann. Intern. Med. 110: 108-114) and in AIDS and cancer patients undergoing chemotherapy (Danna, R P, Rudnick, S A, Abels, R I In: M B, Garnick, ed. Erythropoietin in Clinical Applicationsxe2x80x94An International Perspective. New York, N.Y.: Marcel Dekker;1990: p. 301-324). However, the bioavailability of currently available protein therapeutics such as EPO is limited by their short plasma half-life and susceptibility to protease degradation. These shortcomings prevent them from attaining maximum clinical potency.
Accordingly, the present invention is a new class of PEG derivatives of EPO. The physiologically active PEG-EPO conjugates of this invention comprise an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have the primary structure of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites; said glycoprotein being covalently linked to from one to three lower-alkoxy poly(ethylene glycol) groups, each poly(ethylene glycol) group being covalently linked to the glycoprotein via a linker of the formula xe2x80x94C(O)xe2x80x94Xxe2x80x94Sxe2x80x94Yxe2x80x94 with the C(O) of the linker forming an amide bond with one of said amino groups, X is xe2x80x94(CH2)kxe2x80x94 or xe2x80x94CH2(Oxe2x80x94CH2xe2x80x94CH2)kxe2x80x94, k is from 1 to 10, Y is 
the average molecular weight of each poly(ethylene glycol) moiety is from about 20 kilodaltons to about 40 kilodaltons, and the molecular weight of the conjugate is from about 51 kilodaltons to about 175 kilodaltons.
Compared to unmodified EPO (i.e., EPO without a PEG attached) and conventional PEG-EPO conjugates, the present conjugates have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. The conjugates of this invention have the same uses as EPO. In particular, the conjugates of this invention are useful to treat patients by stimulating the division and differentiation of committed erythroid progenitors in the bone marrow in the same way EPO is used to treat patients.
The present invention also includes a method for the treatment of anemia in a human. The present invention also includes a method for preparing the erythropoietin glycoprotein products which comprises covalently reacting an xcex5-amino group of a lysine amino acid of an erythropoietin protein with a bi-functional reagent to form an intermediate with an amide linkage. The bi-functional reagent contains a reactive group and a protected thiol group. The amide-linked intermediate is then covalently reacted with an activated polyethylene glycol derivative to form the erythropoietin glycoprotein product of the present invention.